Project 1: Coupling of actin polymerization and integrin-mediated adhesion by a molecular clutch in other cell adhesive structures: Phagocytosis. Valentin Jaumouille Goal: Test the hypothesis that integrin-mediated adhesion is coupled to actin polymerization via a molecular clutch in non-lamellipodial actin structures: Phaocytosis by macrophages. In lamellipodia, the force of cortical actin flow is indirectly harnessed via a mechanosensitive molecular clutch to drive integrin activation and cell-ECM traction generation to promote cell migration. However, cortical actin flow and integrin activation are not limited to lamellipodia, but also occur in ventral actin waves or during T-cell/APC interactions. M2 and X2 integrins (complement receptor 3 and 4) are highly expressed in macrophages, neutrophils and dendritic cells, where they are the main phagocytic receptors for many pathogens and participate in clearance of dead and tumor cells. Although it is established that phagocytosis requires intact actin, actin dynamics and their specific contribution to 2 integrin function in phagocytosis is unknown. In addition, it is not known if integrin-mediated phagocytosis is mechanosensitive; i.e. if a fundamental way that macrophages sense self vs non-self or alive vs dead is through integrin-mediated stiffness sensing. The current view in the field of immunology of integrin/complement-mediated phagocytosis by macrophages is that Rho and Dia-mediated actin is required for particle sinking into the cell. However, how the plasma membrane wraps around the particle is not known, and there is no known role for FA proteins or non-receptor tyrosine kinase activity. Although it has been shown that Fc-mediated phagocytosis is mechanosensitive and does not require Arp2/3, whether these properties hold true for integrin-dependent phagocytosis is not known. Innovation/Approach: Our hypothesis that actin polymerization at the plasma membrane is fundamentally coupled to integrin-mediated adhesion by a FA molecular clutch during phagocytosis in macrophages is novel. Together with Eric Betzig, we used high resolution 3D confocal and super-resolution microscopy of immunolocalized endogenous proteins and expressed fluorescent-tagged F-actin, integrins and FA proteins during phagocytosis of complement-opsonized particles by RAW macrophages. We manipulated actin, vinculin, and signaling molecules pharmacologically or via siRNA. 3D super-resolution imaging of fluorescent-tagged actin showed that engagement of particles by macrophages was mediated by actin-filled protrusions that wrapped around the particle, contrasting with the sinking mechanism previously proposed. Analysis of actin dynamics by fluorescent speckle microscopy revealed processive actin assembly at the leading edge of the protruding phagocytic cup with very little retrograde flow relative to the particle, suggesting strong coupling between polymerizing actin and complement-engagedintegrins. Drug perturbation and imaging of the Arp2/3 complex showed that it was required for particle internalization and was localized to the leading edge of the phagocytic protrusion. Immunolocalization and imaging of fluorescent-tagged proteins showed thatintegrins formed small focal complex-like adhesions at the phagocytic cup that contained vinculin, -actinin, zyxin, as well as tyrosine-phosphorylated paxillin, FAK and Syk. Pharmacological perturbation showed that vinculin recruitment to the cup required tyrosine kinase activity, and reduction of vinculin expression by siRNA in frustrated phagocytosis traction force microscopy assays showed that vinculin was required to promote force transmission to complement-engaged integrins. A manuscript is being submitted Project 3:How is adhesion disassembly during mitosis regulated? A hallmark of cell division is the drastic shape change during mitotic cell rounding. Mitotic rounding is required for proper chromosome segregation as well as spindle positioning, orientation and stability, and defects can lead to aneuploidy. A major unanswered question in the cell adhesion field is what triggers mitotic FA disassembly and how important is it. Hawa Racine-Thiam from Matthieu Piels lab has begun to tackle this using live cell imaging of FA, nuclear and cell cycle markers. Her preliminary data suggests that FA loss occurs in prophase before nuclear envelope breakdown and coincides with cyclin B1 nuclear translocation. She is analyzing FA dynamics to see if the rate of FA formation or disassembly are regulated independently, and using inhibitors of mitotic kinases (CDK1, PLK1) to determine how. She is employing traction force microscopy to determine if FAs get torn from the substrate or are passively detached. She plans to test the classic pathways for FA disassembly (FAK, Calpain, clathrin, myosin II) (42,43,140142) and the role of calpain cleavage targets (talin, paxillin, tensin1) (143,144) using point mutants. She has begun testing the role of integrin inactivation in mitotic FA disassembly using Mn2+ and integrin activating antibodies. She found that integrin inactivation is required for mitotic de-adhesion, but quite unexpectedly, that blocking integrin inactivation caused failure of cells to complete mitosis, with a GFP-PCNA (cell cycle marker (145)) localization pattern indicating a G2 block. This suggests an integrin inactivation G2-M checkpoint. We are super-excited about this, and want to check several cell types and integrin family members to be sure. Hawa has also started looking for targets of the mitotic kinases, and has found that paxillin and tensin1 undergo some post-translational modifications specifically at mitosis, yet to be determined by mass spectrometry. Preliminary data indicate that knocking down these proteins using siRNA leads to a delay in mitotic entry and a decrease in rounding time. We will sort this out into two important papers. project 4A.3.2. Mechanism of LIM-domain mechanosensation. We and others found that many LIM domain-containing proteins were depleted from the proteome of FAs isolated under conditions that inhibit cell contractility (38,100). This implies that LIM domains may serve as a modular cytoskeletal/FA tension sensors that could mediate protein localization to these sites in a force-dependent manner to facilitate mechanotransduction. We and Mary Beckerle discovered several years ago that the LIM protein zyxin rapidly accumulates on sites of stress fiber strain/tearing to prevent their breakage by recruiting a repair complex consisting of -actin